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O café é uma das bebidas mais consumidas e um dos principais produtos agrícolas,comercializado mundialmente. A espécie Coffea arabica, preferível pelos agricultores, fornece grãos cuja bebida é reconhecida como de melhor qualidade pois constituí-se bioquimicamente de elevadas concentrações de carboidratos, lipídeos e trigonelina. O desenvolvimento e crescimento do fruto são caracterizados pela evolução dos tecidos embrionários e alterações bioquímicas, ilustrando a importância do seu estudo para melhor compreender as características finais dos grãos. O objetivo deste trabalho foi desenvolver uma nova metodologia eficaz na identifcação do conteúdo protéico expresso no endosperma do fruto do café da espécie C. arabica var. IAPAR59 (I59) em diferentes estágios de maturação. A metodologia utilizada neste trabalho baseia-se na estratégia proteômica bottom-up, com digestão protéica, análises por cromatografia líquida em fase reversa e espectrometria de massa com interfaces on-line e off-line. O primeiro permitiu o assinalamento da estrutura primária completa de vinte e sete peptídeos que foram identificados por alinhamento semântico local (BLAST) e apresentaram homologia com quatro proteínas com função ainda desconhecida e nove proteínas com funções já descritas, dentre elas a globulina 11S e a dehydrina, envolvida na proteção osmótica das células vegetais. A interface on-line permitiu o assinalamento da estrutura primária de trinta e quatro peptídeos, dos quais treze deles correspondiam a proteínas com funções ainda desconhecidas, quatro com sequências ainda não descritas e quatorze proteínas com funções conhecidas expressas nos diferentes estágios de desenvolvimento. Diante do exposto, as técnicas utilizadas neste trabalho abrem boas perspectivas para uma melhor compreenção das alterações proteômicas que ocorrem durante os diferentes estágios de desenvolvimento e maturação dos grãos cafeeiros. Entretanto, ajustes metodológicos ainda se fazem necessários para permitir a identificação de um número maior de proteínas, especialmente aquelas em menor abundância.

O melhoramento da qualidade dos grãos de café com o objetivo de agregar maior valor e aumentar a produtividade é um desafio de longa data. Entretanto, com o desenvolvimento da biologia molecular e da bioquímica permitiu-se um novo nível de melhoramento a partir do conhecimento das moléculas e das vias bioquímicas com influência direta na qualidade e no aroma da bebida. O desenvolvimento do fruto de café é caracterizado por várias mudanças nos tecidos e alterações bioquímicas, ilustrando a importância do seu estudo para melhor compreender as características finais dos grãos de café. O objetivo deste trabalho é desenvolver uma ferramenta eficaz de identifcação do conteúdo protéico expresso nos diferentes estágios de desenvolvimento de frutos da espécie Coffea arabica com possível influência no aroma e sabor da bebida de café. A metodologia utilizada neste trabalho baseia-se na estratégia proteômica do tipo "bottom-up", com digestão protéica e análise por cromatografia líquida de fase reversa e espectrometria de massa em três estágios de maturação de grãos de café variedade arábica: grãos verdes (151 dias após a floração), maduros (210 dias após a floração) e secos (grãos maduros deixados ao ambiente por 13 dias), coletados no ano de 2008. Os dados demonstram que doze proteínas puderam ser identificadas através de buscas de similiaridade feitas em um banco de dados da Embrapa. Embora a metodotologia proposta neste trabalho tenha apresentado uma riqueza de peptídios para análise em cromatografia líquida e MALDI-TOF quando comparada a outras, faz-se necessário a busca de metedologias alternativas, pois a abundância relativa da proteína 11 S constitui um empecilho para a identificação de um maior número de moléculas.

The development of molecular tools for rapid generation of drought-tolerant coffee varieties is amongst the priorities of the Brazilian Research Program on Coffee Genomics. The principal aim of this study was to investigate the molecular mechanisms underlying the response to drought stress in coffee plants by an integrated approach. As part of the Brazilian Coffee EST project, two EST libraries from leaves of drought-stressed plants of Coffea arabica cv. Rubi (drought sensitive) and C. canephora clone 14 (drought tolerant), were generated. The plant material of C. arabica and C. canephora were obtained from field and pot trials, respectively. More than 15,000 clones were sequenced and, after trimming and clustering, resulted in 10,924 reads grouped on 6,141 unigenes (1,779 contigs and 4,362 singlets). The approaches used to identify candidate genes (ESTs) underlying stress responses in coffee consisted of an in silico analysis of ESTs generated from drought-stressed and control libraries, physiological characterization, transcription profiling of drought-stressed and control tissues and protein profiling by 2-DE coupled with tryptic peptide identification by MALDI-TOF-MS/MS. These integrated analysis resulted in the identification of several candidate drought-responsive genes. Results of gene expression (northern-blot and q-PCR) are presented for some GC presenting differential gene expression in leaves and roots of sensitive vs. tolerant clones of C. canephora submitted or not to water limitation. (Texte intégral)

The principal aim of this study was to investigate the molecular mechanisms underlying the response to drought stress in coffee plants by different approaches. In order to indentify candidate genes involved in controlling drought tolerance in coffee plants, different strategies were followed in our laboratories. The first used the nucleic data generated by the Brazilian Coffee EST project to identify candidate genes (CG) by in silico analysis (electronic Northern-blot). Differential expression of these CG was verified in leaves and roots from drought-tolerant and susceptible clones of C. canephora var. Conilon by Northern-blot and quantitative PCR experiments. The second was based on the screening of macroarray membranes spotted with coffee ESTs which were hybridized separately with leaf cDNA probes of the same clones. Finally, 2D gel electrophoresis was also performed to selected proteins presenting differential accumulation in leaves of the same clones. These proteins were analyzed by MALDI-TOF-MS/MS leading to the identification of a new set of CG. Results concerning the identification of CG by these different approaches are presented and discussed.

The quantification of differences in the proteome between physiological states is a challenging technical task. Two-dimensional polyacrylamide gel electrophoresis (2-DE) is still the standard method to separate complex protein mixtures. However, the 2-DE gel approach is insufficient to analyze entire proteomes, including very low abundance proteins. Some problems are overcome by LC/MS-based methods, but mass spectrometry is not inherently quantitative because the peptides can have differences in spectrometric response. In this study, proteins were extracted from leafs and roots of Coffea canephora (Clone 14, drought-tolerant and clone 109A, drought-sensitive) unstressed and submitted to a severe water deficit. Changes in protein profiles and concentrations were analyzed in both genotypes and water regimes. Protein extracts were separated by 2-DE and proteins differentially expressed were identified using MALDI-ToF/ToF mass spectrometry (MS) of tryptic peptides. Trans-Proteomic Pipeline (TPP) was used for analyzing, validating, and storing protein identification data. The X!Tandem software was used for MS/MS ion search against a EST-based database of Coffea. Alternatively, the protein extracts were treated with trypsin and peptides were pre-fractionated by pI before analysis by 2D-LC coupled with a Q-tof mass spectrometer. The results from different experiments were processed and compared by using differential label-free quantitative analysis with the LC-MS imaging based quantification softwares msInspect (http://proteomics.fhcrc.org) and SuperHirn (http://tools.proteomecenter.org). (Texte intégral)

Coffee is an important commodity worldwide with about 125 million people depending on this crop for their livelihoods. Drought stress significantly affects coffee yield with losses estimates in Brazil, varying from 10 to 15 % in recent years. Therefore, the development of molecular tools for rapid generation of drought-tolerant coffee varieties is amongst the priorities of the Brazilian Research Program on Coffee Genomics. The principal aim of this study was to investigate the molecular mechanisms underlying the response to drought stress in coffee plants by an integrated approach. As part of the Brazilian Coffee EST project, two EST libraries from leaves of drought-stressed plants of Coffea arabica cv. Rubi (drought sensitive) and C. canephora clone 14 (drought tolerant), were generated. The plant material of C. arabica and C. canephora were obtained from field and pot trials, respectively. More than 15,000 clones were sequenced and, after trimming and clustering, resulted in 10,924 reads grouped on 6,141 unigenes (1,779 contigs and 4,362 singlets). The approaches used to identify candidate genes (ESTs) underlying stress responses in coffee consisted of an in silico analysis of ESTs generated from drought-stressed and control libraries, physiological characterization, transcription profiling of drought-stressed and control tissues and protein profiling by 2-DE coupled with tryptic peptide identification by MALDI-TOF-MS/MS. These integrated analysis resulted in the identification of several candidate drought-responsive genes. In addition, a mapping population from crosses of C. canephora clones contrasting for this trait is also under way for future association studies and QTL mapping. (Texte intégral)